Throughout this application, citations for various publications are provided within parentheses in the text. The disclosures of these publications are hereby incorporated in their entirety by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The function of carbohydrates as structural materials and as energy storage units in biological systems is well recognized. By contrast, the role of carbohydrates as signaling molecules in the context of biological processes has only recently been appreciated. (M. L. Phillips, E. Nudelman, F. C. A. Gaeta, M. Perez, A. K. Singhal, S. Hakomori, J. C. Paulson, Science, 1990, 250, 1130; M. J. Polley, M. L. Phillips, E. Wagner, E. Nudelman, A. K. Singhal, S. Hakomori, J.C. Paulson, Proc. Natl. Acad. Sci. USA, 1991, 88, 6224: T. Taki, Y. Hirabayashi, H. Ishikawa, S. Kon, Y. Tanaka, M. Matsumoto, J. Biol. Chem., 1986, 261, 3075; Y. Hirabayashi, A. Hyogo, T. Nakao, K. Tsuchiya, Y. Suzuki, M. Matsumoto, K. Kon, S. Ando, ibid., 1990, 265, 8144; O. Hindsgaul, T. Norberg, J. Le Pendu, R. U. Lemieux, Carbohydr. Res., 1982, 109, 109; U. Spohr, R. U. Lemieux, ibid., 1988, 174, 211) The elucidation of the scope of carbohydrate involvement in mediating cellular interaction is an important area of inquiry in contemporary biomedical research. The carbohydrate molecules, carrying detailed structural information, tend to exist as glycoconjugates (cf. glycoproteins and glycolipids) rather than as free entities. Given the complexities often associated with isolating the conjugates in homogeneous form and the difficulties in retrieving intact carbohydrates from these naturally occurring conjugates, the applicability of synthetic approaches is apparent. (For recent reviews of glycosylation see: Paulsen, H., Angew. Chem. Int. Ed. Engl., 1982, 21, 155; Schmidt, R. R., Angew. Chem. Int. Ed. Engl., 1986, 25, 212; Schmidt, R. R., Comprehensive Organic Synthesis, Vol. 6, Chapter 1(2), Pergamon Press, Oxford, 1991; Schmidt, R. R., Carbohydrates, Synthetic Methods and Applications in Medicinal Chemistry, Part I, Chapter 4, VCH Publishers, Weinheim, N.Y., 1992. For the use of glycals as glycosyl donors in glycoside synthesis, see Lemieux, R. U., Can. J. Chem., 1964, 42, 1417; Lemieux, R. U., Faser-Reid, B., Can. J. Chem., 1965, 43, 1460; Lemieux, R. U., Morgan, A. R., Can. J. Chem., 1965, 43, 2190; Thiem, J., Karl, H., Schwentner, J., Synthesis, 1978, 696; Thiem. J. Ossowski, P., Carbohydr. Chem., 1984, 3, 287; Thiem, J., Prahst, A., Wendt, T. Liebigs Ann. Chem., 1986, 1044; Thiem, J. in Trends in Synthetic Carbohydrate Chemistry, Horton, D., Hawkins, L. D., McGarvvey, G. L., eds., ACS Symposium Series #386, American Chemical Society, Washington, D.C., 1989, Chapter 8.)
The carbohydrate domains of the blood group substances contained in both glycoproteins and glycolipids are distributed in erythrocytes, epithelial cells and various secretions. The early focus on these systems centered on their central role in determining blood group specificities. (R. R. Race and R. Sanger, Blood Groups in Man, 6th ed., Blackwell, Oxford, 1975) However, it is recognized that such determinants are broadly implicated in cell adhesion and binding phenomena. (For example, see M. L. Phillips, E. Nudelman, F. C. A. Gaeta, M. Perez, A. K. Singhal, S. Hakomori, J. C. Paulson, Science, 1990, 250, 1130.) Moreover, ensembles related to the blood group substances in conjugated form are encountered as markers for the onset of various tumors. (K. O. Lloyd, Am. J. Clinical Path., 1987, 87, 129; K. O. Lloyd, Cancer Biol., 1991, 2, 421) Carbohydrate-based tumor antigenic factors might find applications at the diagnostic level, as resources in drug delivery or ideally in immunotherapy. (Toyokuni, T., Dean, B., Cai, S., Boivin, D., Hakomori, S., and Singhal, A. K., J. Am. Chem Soc., 1994, 116, 395; Dranoff, G., Jaffee, E., Lazenby, A., Golumbek, P., Levitsky, H., Brose, K., Jackson, V., Hamada, H., Paardoll, D., Mulligan, R., Proc. Natl. Acad. Sci. USA, 1993, 90, 3539; Tao, M-H., Levy, R., Nature, 1993, 362, 755; Boon, T., Int. J. Cancer, 1993, 54, 177; Livingston, P. O., Curr. Opin. Immunol., 1992, 4, 624; Hakomori, S., Annu. Rev. Immunol., 1984, 2, 103; K. Shigeta, et al., J. Biol. Chem., 1987, 262, 1358)
The use of synthetic carbohydrate conjugates to elicit antibodies was first demonstrated by Gobel and Avery in 1929. (Goebel, W. F., and Avery, O. T., J. Exp. Med., 1929, 50, 521; Avery, O. T., and Goebel, W. F., J. Exp. Med., 1929, 50, 533.) Carbohydrates were linked to carrier proteins via the benzenediazonium glycosides. Immunization of rabbits with the synthetic antigens generated polyclonal antibodies. Other workers (Allen, P. Z., and Goldstein, I. J., Biochemistry, 1967, 6, 3029; Rxc3xcde, E., and Delius, M. M., Carbohydr. Res., 1968, 8, 219; Himmelspach, K., et al., Eur. J. Immunol., 1971, 1, 106; Fielder, R. J., et al., J. Immunol., 1970, 105, 265) developed similar techniques for conjugation of carbohydrates to protein carriers. Most of them suffered by introducing an antigenic determinant in the linker itself, resulting in generation of polyclonal antibodies. Kabat (Arakatsu, Y., et al., J. Immunol., 1966, 97, 858), and Gray (Gray, G. R., Arch. Biochem. Biophys. 1974, 163, 426) developed conjugation methods that relied on oxidative or reductive coupling, respectively, of free reducing oligosaccharides. The main disadvantage of these techniques, however, is that the integrity of the reducing end of the oligosaccharide was compromised. In 1975 Lemieux described the use an 8-carbomethoxy-1-octanol linker (Lemieux, R. U., et al., J. Am. Chem. Soc., 1975, 97, 4076) which alleviated the problem of linker antigenicity and left the entire oligosaccharide intact. Equally effective in producing glycoconjugates was the allyl glycoside method described by Bernstein and Hall. (Bernstein, M. A., and Hall, L. D., Carbohydr. Res., 1980, 78, C1.) In this technique the allyl glycoside of the deblocked sugar is ozonized followed by a reductive workup. The resultant aldehyde is then reductively coupled to a protein carrier with sodium cyanoborohydride.
In the mid-70""s and early 80""s Lemieux and his collaborators made contributions to antibody production stimulated by synthetic glycoconjugates (Lemieux, R. U., et al., J. Am. Chem. Soc., 1975, 97, 4076) and to conformational issues (Lemieux, R. U., et al., Can. J. Chem., 1979, 58, 631; Spohr, U., et al., Can. J. Chem., 1985, 64, 2644; Vandonselaar, M., et al., J. Biol. Chem., 1987, 262, 10848) important in the interactions of the blood group determinants (and analogues thereof) with the carbohydrate binding proteins known as lectins. More recently, workers at Bristol-Meyers Squibb reported the X-ray crystal structure of the Lewisy epitope complexed with the antibody BR96. (Jeffrey, P. D., et al., Nature Structural Biol., 1995, 2, 466.) Two main components appear to govern recognition between carbohydrates and most antibodies. The first is multiple hydrogen bonding between the sugar hydroxyls and the amino acid residues of Asp, Asn, Glu, Gln, and Arg. The second major interaction is stacking between the sugar-ring faces and aromatic side chains, which occurs most frequently with tryptophan. In the complex with BR96 the most significant interactions involve the latter; additional hydrogen bonding occurs between the sugar hydroxyls and the indole nitrogens. Most antibody binding sites can support about 6 linear carbohydrate residues in a groove or cavity shaped binding site.
Glycoconjugates would be used, ideally, in direct immunotherapy or the monoclonal antibodies generated from vaccinations could be used to specifically target known chemotherapeutic agents to tumor sites. The immune response to carbohydrates is generally not strong, resulting mainly in production of IgM type antibodies. IgM antibodies are capable of complement fixation. Complement is a family of enzymes that can lyse cells to which antibodies are bound. The response to carbohydrate antigens normally does not enlist the use of T-cells which would aid in the body""s rejection of the tumor. While the probability of complete tumor rejection as a result of vaccination with a conjugate is unlikely, such treatments will boost immune surveillance and recurrence of new tumor colonies can be reduced. (Dennis, J., Oxford Glycosystems Glyconews Second, 1992; Lloyd, K. O., in Specific Immunotherapy of Cancer with Vaccines, 1993, New York Academy of Sciences, 50-58.) Toyokuni and Singhal have described a synthetic glycoconjugate (Toyokuni, T., et al., J. Am. Chem. Soc., 1994, 116, 395) that stimulated a measurable IgG titer, a result which is significant since an IgG response is generally associated with enlistment of helper T cells.
The use of immunoconjugates has shown promise in the reduction of large tumor masses. The workers at Bristol-Meyers Squibb (Trail, P. A., et al., Science, 1993, 261, 212) have described the attachment of the known chemotherapeutic drug doxorubicin to the antibody BR96. BR96 is an anti-Lewisy antibody which has been shown to bind to human breast, lung and colon carcinomas. Athymic mice that have had human cancers (L2987-lung, RCA-colon, and MCF7-breast carcinomas) xenografted subcutaneously were treated with the drug-antibody conjugate (BR96-DOX). The result was complete regression of the tumor mass in 78% of the mice treated. BR96 is efficiently internalized by cellular lysosomes and endosomes following attachment to the cell surface. The change in pH upon internalization results in cleavage of the labile hydrazone thereby targeting the drug specifically to the desired site.
Many of the blood group determinant structures can also occur in normal tissues. Antigen expression in normal cells and cancer cells can have subtle distributional differences. In the case of Ley, (which does appear in normal tissues) the expression of the determinant in tumor cells tends to be in the form of mucins which are secreted. Mucins are glycoproteins with a high content of the amino acids serine and threonine. It is through the hydroxyl functionality of these amino acids that Lewisy is linked. Thus, in terms of generating competent antibodies against tumor cells expressing the Ley antigen it is important that the antibody recognize the mucin structure.
Structurally, the blood group determinants fall into two basic categories known as type I and type II. Type I is characterized by a backbone comprised of a galactose 1-3b linked to N-acetyl glucosamine while type II contains, instead, a 1-4b linkage between the same building blocks (cf. N-acetyl lactosamine). The position and extent of a-fucosylation of these backbone structures gives rise to the Lewis-type and H-type specificities. Thus, monofucosylation at the C4-hydroxyl of the N-acetyl glucosamine (Type I series) constitutes the Lea type, whereas fucosylation of the C3-hydroxyl of this sugar (Type II series) constitutes the Lex determinant. Additional fucosylation of Lea and Lex types at the C2,-hydroxyl of the galactose sector specifies the Leb and Ley types, respectively. The Ley determinant is expressed in human colonic and liver adenocarcinomas. (Levery, S. B., et al., Carbohydr. Res., 1986, 151, 311; Kim, Y. S., J. Cellular Biochem. Suppl., 16G 1992, 96; Kaizu, T., et al., J. Biol. Chem., 1986, 261, 11254; Levery, S. B., et al., Carbohydr. Res. 1986, 151, 311; Hakomori, S., et al., J. Biol. Chem., 1984, 259, 4672; Fukushi, Y., et al., ibid., 1984, 259, 4681; Fukushi, Y., et al., ibid., 1984, 259, 10511.)
The presence of an a-monofucosyl branch, solely at the C2,-hydroxyl in the galactose moiety in the backbone, constitutes the H-type specifity (Types I and II). Further permutation of the H-types by substitution of a-linked galactose or a-linked N-acetylgalactosamine at its C3,-hydroxyl group provides the molecular basis of the familiar serological blood group classifications A, B, and O. (Lowe, J. B., The Molecular Basis of Blood Diseases, Stamatoyannopoulos, et. al., eds., W. B. Saunders Co., Philadelphia, Pa, 1994, 293.)
Several issues merit consideration in contemplating the synthesis of such blood group substances and their neoglycoconjugates. For purposes of synthetic economy it would be helpful to gain relief from elaborate protecting group manipulations common to traditional syntheses of complex branched carbohydrates. Another issue involves fashioning a determinant linked to a protein carrier. It is only in the context of such conjugates that the determinants are able to galvanize B-cell response and complement fixation. In crafting such constructs, it is beneficial to incorporate appropriate spacer units between the carbohydrate determinant and the carrier. (Stroud, M. R., et al., Biochemistry, 1994, 33, 10672; Yuen, C.-T., et al., J. Biochem., 1994, 269, 1595; Stroud, M. R., et al., J. Biol. Chem., 1991, 266, 8439.)
The present invention provides new strategies and protocols for oligosaccharide synthesis. The object is to simplify such constructions such that relatively complex domains can be assembled with high stereo-specifity. Major advances in glycoconjugate synthesis require the attainment of a high degree of convergence and relief from the burdens associated with the manipulation of blocking groups. Another requirement is that of delivering the carbohydrate determinant with appropriate provision for conjugation to carrier proteins or lipids. (Bernstein, M. A., and Hall, L. D., Carbohydr. Res., 1980, 78, Cl; Lemieux, R. U., Chem. Soc. Rev., 1978, 7, 423; R. U. Lemieux, et al., J. Am. Chem. Soc., 1975, 97, 4076) This is a critical condition if the synthetically derived carbohydrates are to be incorporated into carriers suitable for biological application.
Antigens which are selective or ideally specific for cancer cells could prove useful in fostering active immunity. (Hakomori, S., Cancer Res., 1985, 45, 2405-2414; Feizi, T., Cancer Surveys, 1985, 4, 245-269) Novel carbohydrate patterns are often presented by transformed cells as either cell surface glycoproteins or as membrane-anchored glycolipids. In principle, well chosen synthetic glycoconjugates which stimulate antibody production could confer active immunity against cancers which present equivalent structure types on their cell surfaces. (Dennis, J., Oxford Glycosystems Glyconews Second, 1992; Lloyd, K. O., in Specific Immunotherapy of Cancer with Vaccines, 1993, New York Academy of Sciences pp. 50-58) Chances for successful therapy improve with increasing restriction of the antigen to the target cell. A glycosphingolipid was isolated by Hakomori and collaborators from the breast cancer cell line MCF-7 and immunocharacterized by monoclonal antibody MBr1-(Bremer, E. G., et al., J. Biol. Chem., 1984, 259, 14773-14777; Menard, S., et al., Cancer Res., 1983, 43, 1295-1300) The novel glycosphingolipid structure 1b (FIG. 8) was proposed for this breast tumor-associated antigen on the basis of methylation and enzymatic degradation protocols. A 1H NMR spectrum consistent with but not definitive for the proposed structure was obtained from trace amounts of isolated antigen. While individual sectors of the proposed structure were not unknown, the full structure was first described based on studies on the breast cancer line. It should be noted that MBr1 also binds to normal human mammary gland tissue and ovarian cancer cell lines. Therefore, 1b as a total entity is likely not restricted to the transformed breast cells. Alternatively, smaller subsections of 1b are adequate for antibody recognition and binding. (The synthesis of the DEF fragment of 1b has been reported, and has been shown to bind to MBr1: Lay, L., et al., Helv. Chim. Acta, 1994, 77, 509-514.)
The compounds prepared by processes described herein are antigens useful in adjuvant therapies as vaccines capable of inducing antibodies immunoreactive with epithelial carcinomas, for example, human colon, lung and ovarian tumors. Such adjuvant therapies have potential to reduce the rate of recurrence of cancer and increase survival rates after surgery. Clinical trials on 122 patents surgically treated for AJCC stage III melanoma who were trated with vaccines prepared from melanoma differentiation antigen GM2 (another tumor antigen which like MBr1 is a cell surface carbohydrate) demonstrated in patients (lacking the antibody prior to immunization) a highly significant increase in disease-free interval (P. O. Livingston, et al., J. Clin Oncol., 12, 1036 (1994))
The present invention provides a method of synthesizing Ley-related antigens as well as artificial protein-conjugates of the oligosaccharide which might be more immunogenic than the smaller glycolipid. The antigen contains a novel array of features including the xcex1-linkage between the B and the C entities, as well as the xcex2-linked ring D gal-NAc residue. (For the synthesis of a related structure (SSEA-3) which lacks the fucose residue see: Nunomura, S.; Ogawa, T., Tetrahedron Lett., 1988, 29, 5681-5684.) The present invention also provides a total synthesis of 1b, rigorous proof that the Hakomori antigen does, in fact, correspond to 1b and the synthesis of a bioconjugatable version of 1b. The conciseness of the synthesis reflects the efficiency of glycal assembly methods augmented by a powerful method for sulfonamidoglycosylation (see, e.g., the transformation of 14b -15b, FIG. 10).